Methamphetamine (METH) use disorder is linked to dire health and societal consequences. Effective treat- ments for METH addiction are not available. METH interacts with the vesicular monoamine transporter-2 (VMAT2), promoting dopamine (DA) release into the cytosol and reversal of the DA transporter to increase ex- tracellular DA, resulting in abuse liability. This project focuses on VMAT2 as a novel therapeutic target, with the overall goal of obtaining a treatment for METH use disorder. Phase 1b studies with lobeline, the initial lead, were completed; however, bitter taste and multiple daily doses were expected to reduce compliance. Lobeline was modified to obtain GZ-793A, which had the desired pharmacology, but also had hERG toxicity. We next identified novel small molecules, GZ-11608 and JPC-077, with greatly reduced hERG interaction, and with po- tent and selective inhibition of VMAT2 function and METH-evoked DA release. These new leads specifically decrease METH self-administration and METH-induced reinstatement in rats at doses that do not alter food reinforcement, suggesting efficacy against relapse. Tolerance does not develop upon repeated dosing. These analogs protect against or do not exacerbate METH-induced striatal DA neurotoxicity. Despite this favorable pharmacologic profile, these analogs have low oral bioavailability. Thus, this proposal focuses on the discov- ery/development of optimized GZ-11608 and JPC-077 analogs with increased oral bioavailability. First, we will substitute deuterium for hydrogen at sites of metabolic liability. If deuterium substitution is not successful, we will use rational design to synthesize a focused library based on each lead. Chemoinformatics, incorporating refined models based on our library of ~520 VMAT2-targeted compounds, was used to predict optimized ana- logs with decreased metabolic liability and desirable drug-like properties. Each analog, from deuterium substi- tution and medchem approaches, will be synthesized and evaluated for selective inhibition of VMAT2, as well as metabolism. We will then assess the pharmacokinetics (PK) to determine oral bioavailability, plasma and brain concentrations and estimate PK parameters following IV, PO and SC dosing. Metabolites exceeding 10% will be synthesized and evaluated for toxicity and potential use as active pharmaceutical ingredient. We will then determine the dose-related inhibition of METH self-administration and reinstatement. Development of tol- erance following repeated administration will be determined. We will conduct abbreviated toxicology studies and assess the potential for off-target interactions and CYP450 inhibition/induction. We will relate the pharma- cokinetics of optimized analogs to pharmacodynamic effects, and determine the maximum tolerated dose, ther- apeutic index and behavioral specificity. Completion of this optimization program will allow for immediate tran- sition of our leads into IND-enabling studies. Successful completion of the project should have a tremendously beneficial health and socioeconomic impact on society, since currently there are no available treatments for METH use disorder.